Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout

Background: The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). Methods: To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses. Results: LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha. Conclusions In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction.

Decrease in luteinizing hormone pulse frequency during a five-hour peripheral Ghrelin infusion in the ovariectomized rhesus monkey

RESUME : La ghrelin est un peptide sécrété par l'estomac jouant un rôle important dans le maintien de l'homéostasie énergétique. Ses taux plasmatiques sont augmentés durant des périodes prolongées de déficit nutritionnel. Une carence énergétique étant souvent associée à une inhibition de l'axe hypothalamo-hypophyso-ovarien, nous avons postulé que l'augmentation des taux circulant de ghrelin pourrait diminuer l'activité du générateur hypothalamique de pulsations de GnRH. Le protocole expérimental impliquait des singes rhésus adultes ovariectomisés (n=6) qui dans un premier temps recevaient durant 3 heures une perfusion de solution saline physiologique afin de mesurer la sécrétion pulsatile de LH à l'état basai. L'expérience se poursuivait alors durant 5 heures par une perfusion intraveineuse de ghrelin humaine (un bolus de 100-150µg suivi par 100-150µg/h) ou le maintien de la perfusion de solution saline physiologique. Des échantillons de sang étaient prélevés toutes les 15 minutes. La perfusion de ghrelin a augmenté ses taux plasmatiques de 2.9 fois par rapport aux valeurs de base. L'administration de ghrelin a significativement diminué la fréquence des pulsations de LH (de 0.89±0.07/h à l'état basai à 0.57±0.10/h durant la perfusion de ghrelin; p<0.05, moyenne±SEM), alors que la fréquence des pulsations de LH est restée inchangée durant la perfusion de solution physiologique. L'amplitude des pulsations de LH n'a pas été modifiée. La ghrelin a également stimulé de manière significative la sécrétion de cortisol et d'hormone de croissance, mais n'a toutefois pas eu d'effet sur la sécrétion de leptin. En conclusion, la ghrelin peut inhiber l'activité du générateur de pulsations de GnRH et pourrait ainsi contribuer à l'inhibition de l'axe de la reproduction observée durant des périodes de carence nutritionnelle, comme notamment chez les patientes souffrant d'anorexie mentale. La ghrelin peut également activer l'axe hypothalamo-hypophyso-surrénalien. Le lien dans cette situation entre l'activation de l'axe surrénalien et l'inhibition de l'axe de la reproduction reste à démontrer. ABSTRACT: Ghrelin, a nutrition-related peptide secreted by the stomach, is elevated during prolonged food deprivation. Because undernutrition is often associated with a suppressed reproductive axis, we have postulated that increasing peripheral ghrelin levels will decrease the activity of the GnRH pulse generator. Adult ovariectomized rhesus monkeys (n = 6) were subjected to a 5-h iv human ghrelin (100- to 150µg bolus followed by 100-150 µg/h) or saline infusion, preceded by a 3-h saline infusion to establish baseline pulsatile LH release. Blood samples were collected at 15-min intervals throughout the experiment. Ghrelin infusion increased plasma ghrelin levels 2.9-fold of baseline. Ghrelin significantly decreased LH pulse frequency (from 0.89 ± 0.07/h in baseline to 0.57 ± 0.10/h during ghrelin infusion; P<0.05, mean ± SEM), whereas LH pulse frequency remained unchanged during saline treatment. LH pulse amplitude was not affected. Ghrelin also significantly stimulated both Cortisol and GH release, but had no effect on leptin. We conclude that ghrelin can inhibit GnRH pulse activity and may thereby mediate the suppression of the reproductive system observed in conditions of undernutrition, such as in anorexia nervosa. Ghrelin also activates the adrenal axis, but the relevance of this to the inhibition of GnRH pulse frequency remains to be established.

Sexual dimorphism in a dioecious population of the wind-pollinated herb Mercurialis annua: the interactive effects of resource availability and competition.

Background and Aims Male-biased sex allocation commonly occurs in wind-pollinated hermaphroditic plants, and is often positively associated with size, notably in terms of height. Currently, it is not well established whether a corresponding pattern holds for dioecious plants: do males of wind-pollinated species exhibit greater reproductive allocation than females? Here, sexual dimorphism is investigated in terms of life history trade-offs in a dioecious population of the wind-pollinated ruderal herb Mercurialis annua.Methods The allocation strategies of males and females grown under different soil nutrient availability and competitive (i.e. no, male or female competitor) regimes were compared.Key Results Male reproductive allocation increased disproportionately with biomass, and was greater than that of females when grown in rich soils. Sexual morphs differentially adjusted their reproductive allocation in response to local environmental conditions. In particular, males reduced their reproductive allocation in poor soils, whereas females increased theirs, especially when competing with another female rather than growing alone. Finally, males displayed smaller above-ground vegetative sizes than females, but neither nutrient availability nor competition had a strong independent effect on relative size disparities between the sexes.Conclusions Selection appears to favour plasticity in reproductive allocation in dioecious M. annua, thereby maintaining a relatively constant size hierarchy between sexual morphs. In common with other dioecious species, there seems to be little divergence in the niches occupied by males and females of M. annua.

Preferential reproduction mode of hermaphrodite papaya plant (Carica papaya L; Caricaceae)

This research was done to study the reproductive system of papaya hermaphrodite plant based on the histochemical nature of pollen grain, stigma receptivity, in vivo pollen grain germination and pollen:ovule ratio. In the histochemical analysis, pollen grains were stained by using Sudan IV and I2KI solution ; the stigma receptivity was assessed by alpha-naphthtyl acetate solution in closed and opened flowers. Pollen germination and pollen tube growing were examined in flower buds near anthesis with 0.1% aniline blue. To estimate the pollen:ovule ratio , anthers from each flower bud were dissected and all pollen grains were counted; ovules were dissected from ovaries and were counted under stereomicroscope. The results indicated that papaya pollen grains are of lipidic nature; the stigmas were receptive before the opening and until 48 hours after opening; the pollen grains germinated and emitted polinic tube before flower opening and the pollen:ovule ratio indicated the predominance of autogamous reproductive system. These results indicate that hermaphrodite papaya trees is preferentially of optional autogamous with cleistogamy.

Regulating Mechanisms of Gonadal and Pituitary Tumorigenesis in Mice Producing Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are structurally and functionally similar glycoprotein hormones acting through the same luteinizing hormone chorionic gonadotropin receptor (LHCGR). The functions of LH in reproduction and hCG in pregnancy are well known. Recently, the expression of LHCGR has been found in many nongonadal tissues and cancers, and this has raised the question of whether LH/hCG could affect the function or tumorigenesis of these nongonadal tissues. We have also previously generated an hCG expressing mouse model presenting nongonadal phenotypes. Using this model it is possible to improve our understanding of nongonadal action of highly elevated LH/hCG. In the current study, we analyzed the effect of moderately and highly elevated hCG levels on male reproductive development and function. The main finding was the appearance of fetal Leydig cell (FLC) adenomas in prepubertal males. However, the development and differentiation of FLCs were not significantly affected. We also show that the function of hCG is different in FLCs and in adult Leydig cells (ALC), because in the latter cells hCG was not able to induce tumorigenesis. In FLCs, LHCGR is not desensitized or downregulated upon ligand binding. In this study, we found that the testicular expression of two G protein-coupled receptor kinases responsible for receptor desensitization or downregulation is increased in adult testis. Results suggest that the lack of LHCGR desensitization or downregulation in FLCs protect testosterone (Te) synthesis, but also predispose FLCs for LH/hCG induced adenomas. However, all the hCG induced nongonadal changes observed in male mice were possible to explain by the elevated Te level found in these males. Our findings indicate that the direct nongonadal effects of elevated LH/hCG in males are not pathophysiologically significant. In female mice, we showed that an elevated hCG level was able to induce gonadal tumorigenesis. hCG also induced the formation of pituitary adenomas (PA), but the mechanism was indirect. Furthermore, we found two new potential risk factors and a novel hormonally induced mechanism for PAs. Increased progesterone (P) levels in the presence of physiological estradiol (E2) levels induced the formation of PAs in female mice. E2 and P induced the expression and nuclear localization of a known cell-cycle regulator, cyclin D1. A calorie restricted diet was also able to prevent the formation of PAs, suggesting that obesity is able to promote the formation of PAs. Hormone replacement therapy after gonadectomy and hormone antagonist therapy showed that the nongonadal phenotypes observed in hCG expressing female mice were due to ovarian hyperstimulation. A slight adrenal phenotype was evident even after gonadectomy in hCG expressing females, but E2 and P replacement was able to induce a similar phenotype in WT females without elevated LH/hCG action. In conclusion, we showed that the direct effects of elevated hCG/LH action are limited only to the gonads of both sexes. The nongonadal phenotypes observed in hCG expressing mice were due to the indirect, gonadal hormone mediated effects of elevated hCG. Therefore, the gonads are the only physiologically significant direct targets of LHCGR signalling.

Desreguladores endócrinos no meio ambiente: efeitos e conseqüências

There is an increasing interest in micropollutants in the environment that can interfere with the endocrine system, affecting health, growth and reproduction of animals and humans. These substances are known as Endocrine Disrupting Chemicals (EDCs) and can be found in domestic sewage, domestic wastewater treatment plant effluents, and in natural and potable waters. There are numerous chemicals classified as EDCs, such as pesticides, chemicals used and produced by chemical industries and natural and synthetic estrogens. EDCs can be related to the increase of the incidence of anomalies in the reproductive system of animals, cancer in humans and reduction of the masculine fertility.

Analyses of the development and glycoproteins present in the ovarian follicles of Poecilia vivipara (Cyprinodontiformes, Poeciliidae)

The morphofunctional aspects of oogenesis of Poecilia vivipara were studied aiming to understand the reproductive biology and development of species with internal fertilization, particularly those belonging to the family Poeciliidae. The stages of gonadal maturation and follicular development were characterized using mesoscopic, histological, histochemical, and lectin cytochemical analyses. Through mesoscopic evaluation the ovarian development was classified in six phases of development: immature, in maturation I, in maturation II, mature I, mature II, and post-spawn. Based on microscopic examination of the ovaries, we identified the presence of oocytes types I and II during the previtellogenic phase and types III, IV, and V during the vitellogenic phase. As oogenesis proceeded the oocyte cytosol increased in volume and presented increased cytoplasmic granule accumulation, characterizing vitellogenesis. The zona radiata (ZR) increased in thickness and complexity, and the follicular epithelium, which was initially thin and consisting of pavimentous cells, in type III oocytes exhibited cubic simple cells. The histochemical and cytochemical analyses revealed alterations in the composition of the molecular structures that form the ovarian follicle throughout the gonadal development. Our study demonstrated differences in the female reproductive system among fish species with internal and external fertilization and we suggest P. vivipara can be used as experimental model to test environmental toxicity.

Occurrence of Toxoplasma gondii DNA in sheep naturally infected and slaughtered in abattoirs in Pernambuco, Brazil

The aim of the present study was to assess the occurrence of antibodies to Toxoplasma gondii and to detect genomic DNA of the parasite in the reproductive organs, fetuses and fetal membranes of sheep in slaughterhouses in the state of Pernambuco, Brazil. The Indirect Immunofluorescence technique (IFA) was used for screening. The Polymerase Chain Reaction (PCR) was used to detect DNA of T. gondii in the animals that were positive in the serology. In the serology, 13/50 samples were positive and genomic DNA of T. gondii was detected in one uterus, tube, ovary, placenta and fetus (heart, brain and umbilical cord) sample from a sheep that was positive in the serology. The present study provides evidence of the occurrence of T. gondii DNA in the organs of the reproductive system, placenta and fetus of a naturally infected sheep.

Biologia floral e sistema de reprodução de Jacquemontia multiflora (Choisy) Hallier f. (Convolvulaceae)

Aspectos da fenologia, biologia da polinização e reprodução de Jacquemontia multiflora foram estudados na Fazenda Catalunha, Santa Maria da Boa Vista-PE. J. multiflora é uma liana anual, que apresenta floração do tipo cornucópia, com pico desta fenofase no bimestre março/abril, que corresponde ao final da estação chuvosa. As flores estão reunidas em cimeiras que apresentam eixo principal desenvolvido, expondo as flores acima da folhagem. As flores são raso-campanuladas, azuis, inodoras e secretam pequenas quantidades de néctar. A antese é diurna, ocorrendo por volta da 5:30h., e a duração das flores é de aproximadamente nove horas, podendo ser consideradas como efêmeras. Abelhas Apidae e Halictidae são os visitantes mais frequentes. Apis mellifera e Trigona spinipes são consideradas como principais polinizadores desta espécie. Quanto ao sistema de reprodução J. multiflora é autógama facultativa, produzindo frutos e sementes por autopolinização manual (30%) e também por polinização cruzada (60%).

Morphology, reproduction and development of Hypoglossum hypoglossoides (Stackhouse) Collins & Hervey (Ceramiales, Rhodophyta) from the south and southeastern Brazilian coast

Hypoglossum hypoglossoides (Stackhouse) Collins & Hervey is reported for the first time from the infralittoral of São Paulo and Santa Catarina states. The species was already reported to the states of Rio de Janeiro, Espírito Santo and Bahia as Hypoglossum tenuifolium (Harvey) J. Agardh var. carolinianum Williams. A detailed description of the morphology and reproduction is given based on field-collected material. Unialgal cultures were initiated from tetraspore germination, and growth rates of gametophytes were determined under different conditions of temperature, photoperiod and irradiance. Gametophytes grew well between 15 to 30 ºC, 14L:10D and 10L:14D photoperiods and irradiance from 20 to 120 µmol photons.m-2.s-1, but presented low percentage of fertile plants in low temperature (15 ºC). Gametophytes cultured in laboratory developed only male reproductive structures. Physiological responses of H. hypoglossoides indicate that the species is well adapted to temperature and light variations which could explain its range of vertical (6-26 m depth) and latitudinal distribution (from Fernando de Noronha to Santa Catarina) as well as the absence of sexual reproduction in the southern limit of its distribution.

Self-, cross- and interspecific pollinations in Passiflora capsularis and P. rubra

This study aimed to characterize the reproductive system of Passiflora capsularis L. and P. rubra L. In vivo controlled pollinations, in vitro pollen germination and pollen-ovule (P:O) ratio evaluation were conducted. In self-pollination, intraspecific and interspecific pollination, P. capsularis showed means of 62.5, 68.7 and 48.4% of fertilized flowers, while in P. rubra, the averages were 67.2, 62.5 and 46.9%, respectively. For in vitro germination, 52.2% of P. capsularis pollen grains germinated while in P. rubra, the percentage was 64.4. The P:O ratio was 22.4 for P. capsularis, and 27.4 for P. rubra, which included them in the category of obligatory autogamous. Passiflora capsularis and P. rubra can reproduce both by self-pollination and cross-pollination, and crossings between the two species succeeded though the success rate was lower than 50%. The characteristics of the reproductive system of both species allow the use of greater range of options on breeding methods for production of ornamental Passiflora plants.

Is the infertility in hypothyroidism mainly due to ovarian or pituitary functional changes?

The objective of the present study was to examine whether hypothyroidism affects the reproductive system of adult female rats by evaluating ovarian morphology, uterus weight and the changes in serum and pituitary concentrations of prolactin and gonadotropins. Three-month-old female rats were divided into three groups: control (N = 10), hypothyroid (N = 10), treated with 0.05% 6-propyl-2-thiouracil (PTU) in drinking water for 60 days, and T4-treated group (N = 10), receiving daily sc injections of L-thyroxine (0.8 µg/100 g body weight) during the last 10 days of the experiment. At the end of 50 days of hypothyroidism no hypothyroid animal showed a regular cycle, while 71% of controls as well as the T4-treated rats showed regular cycles. Corpora lutea, growing follicles and mature Graafian follicles were found in all ovaries studied. The corpora lutea were smaller in both the hypothyroid and T4-replaced rats. Graafian follicles were found in 72% of controls and only in 34% of hypothyroid and 43% of T4-treated animals. Serum LH, FSH, progesterone and estradiol concentrations did not differ among the three groups. Serum prolactin concentration and the pituitary content of the three hormones studied were higher in the hypothyroid animals compared to control. T4 treatment restored serum prolactin concentration to the level found in controls, but only partially normalized the pituitary content of gonadotropins and prolactin. In conclusion, the morphological changes caused by hypothyroidism can be a consequence of higher prolactin production that can block the secretion and action of gonadotropins, being the main cause of the changes observed.

Ghrelin attenuates the growth of HO-8910 ovarian cancer cells through the ERK pathway

Ovarian cancer is one of the most common causes of death from gynecologic tumors and is an important public health issue. Ghrelin is a recently discovered bioactive peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR). Several studies have identified the protective effects of ghrelin on the mammalian reproductive system. However, little research has been done on the effects of ghrelin on ovarian cancer cells, and the underlying mechanisms of these effects. We sought to understand the potential involvement of mitogen-activated protein kinases (MAPKs) in ghrelin-mediated inhibition of growth of the ovarian line HO-8910. We applied different concentrations of ghrelin and an inhibitor of the ghrelin receptor (D-Lys3-GHRP-6) to HO-8910 cells and observed the growth rate of cells and changes in phosphorylation of the MAPKs ERK1/2, JNK and p38. We discovered that ghrelin-induced apoptosis of HO-8910 cells was though phosphorylated ERK1/2, and that this phosphorylation (as well as p90rsk phosphorylation) was mediated by the GHSR. The ERK1/2 pathway is known to play an essential part in the ghrelin-mediated apoptosis of HO-8910 cells. Hence, our study suggests that ghrelin inhibits the growth of HO-8910 cells primarily through the GHSR/ERK pathway.

Family rivalry in the testis: Retinoblastoma protein and E2F transcription factors in testicular development and adult spermatogenesis

Disorders of male reproductive health are becoming increasingly prevalent globally. These defects, ranging from decreasing sperm counts to an increasing rate of infertility and testicular cancer, have a common origin in the early phases of testicular development, but the exact mechanisms that cause them remain unknown. Testicular development and adult spermatogenesis are complex processes in which different cell types undergo mitosis, meiosis, differentiation and apoptosis. The retinoblastoma protein family and its associated E2F transcription factors are key regulators of these cellular events. In the present study, the functions of these factors in postnatal testicular development and adult spermatogenesis were explored using different animal models. In addition, a new application of flow cytometry to study testicular cell dynamics was developed. An ablation of retinoblastoma protein in mouse Sertoli cells resulted in their cell cycle re-entry in adult testes, dedifferentiation and a severe spermatogenic defect. We showed that deregulated E2F3 contributed to these changes. Our results indicated that the E2F1 transcription factor is critical for the control of apoptosis in the developing postnatal testis. In the adult testis, E2F1 controls the maintenance of the spermatogonial stem cell pool, in addition to inhibiting apoptosis of spermatocytes. In summary, this study elucidated the complex interdependencies of the RB and E2F transcription factor families in the control of postnatal testicular development and adult spermatogenesis. Furthermore, this study provided a new methodology for the analysis of testicular cells.

Analyse de la fertilité des vaches laitières Holstein «Repeat Breeder»

L’importance de la problématique des vaches laitières non gestantes avec multiples inséminations (› 3) communément appelées « Repeat Breeder » (RB), au Québec, repose sur deux facteurs: son incidence et son impact économique. Actuellement, l’incidence du syndrome RB au Québec est de ± 25% (Rapport annuel, juin 2008, www.dsahr.ca). Les pertes monétaires associées à ce problème proviennent des frais vétérinaires et d’insémination, de la diminution de productivité ainsi que des pertes en lien avec la réforme involontaire. Afin d’avoir de meilleures connaissances sur ce syndrome, nous devons connaître les facteurs de risques généraux impliqués et ensuite explorer la condition individuelle de ces vaches problèmes. Dans la première partie de ce mémoire, une banque de données informatisées répertoriant 418 383 lactations fut analysée. L’objectif de ce projet de recherche était d’évaluer l’impact des problèmes reproducteurs post-partum et l’effet du nombre de lactations comme facteurs de risques de la vache Repeat Breeder. L’analyse a permis d’établir la dystocie comme étant la condition ayant le plus de conséquences sur la fertilité future. D’autres facteurs de risques à savoir le nombre de lactations influencent le pronostic reproducteur. La deuxième partie de ce mémoire consistait à explorer, à l’aide d’outils cliniques, la condition individuelle des vaches RB. Une étude cohorte fut menée sur des vaches Holstein en fin de période d’attente volontaire au jour 7 du cycle oestral. Les tests cliniques étudiés furent la vaginoscopie, l’examen transrectal, l’ultrasonographie du système reproducteur, la présence d’estérases leucocytaires, la bactériologie et la biochimie du liquide utérin, la cytologie endométriale et le profil de progestérone sérique. Les résultats de ces tests cliniques dévoilent que l’examen bactériologique du liquide utérin est révélateur du statut reproducteur futur.